This invention relates to a method and apparatus for the fabrication of autologous blood vessels without smooth muscle cells or non-biological scaffolding materials. The principal use of such blood vessels is for small diameter bypass grafts such as coronary artery bypass graft (CABGs), peripheral bypass grafts, or arteriovenous shunts.
Despite dramatic declines in the coronary heart disease related deaths over the last 30 years, heart disease remains the number one cause of death in the industrialized world. The decline in the death rate has coincided with the development and success of CABGs. In this procedure, vessels are typically harvested from the patient's own internal mammary arteries or saphenous veins (called an “autologous graft”) and grafted into the coronary circulation to bypass arteries occluded by atherosclerotic plaques.
One important limitation of this procedure is that grafted vessels are susceptible to plaque regrowth leading to blockages (restenosis). Most CABGs using internal mammary arteries remain functional for 10–12 years. Beyond this timeframe, restenosis rates increase dramatically. Many patients therefore require a second bypass. In 1995, 573,000 grafts were performed in the U.S. alone, and 43% of these were in patients between 45 and 64 years of age, suggesting that a large fraction of these younger patients will live long enough to require a second procedure. However, due to previous vessel harvest or to vascular disease progression, suitable vessels for further arterial reconstruction often are not available. Currently, there is no clinically viable long-term treatment strategy for these patients. Over 50,000 Americans die annually due to the lack of graftable vessels. This number is likely to continue to rise, lagging 10 years behind the exponential growth in primary CABG procedures.
Prior attempts at finding alternate vessel sources focused on synthetic materials or dried and fixed tissue transplants. Larger diameter synthetic grafts have demonstrated reasonable functionality in the peripheral circulation. In the small diameter (less than 6 mm inside diameter) coronary circulation, however, synthetic grafts do not work and typically fail due to thrombogenesis (the formation of blood clots that occlude the vessel). Moreover, the synthetic materials often initiate chronic inflammatory responses that may be the cause of vessel failure.
In an effort to improve synthetic graft integration, synthetic biomaterials have been combined with autologous tissues from the patient. One procedure employs a tubular silicone mandrel surrounded by a Dacron mesh. This was implanted beneath the skin in patients scheduled for vascular reconstruction. After two months, the implant was removed from the patient and the Dacron mesh, along with a tightly integrated fibrous tissue, was slid away from the mandrel. This Dacron-supported scar tissue was then utilized as a vascular graft in the same patient. Although this graft combined autologous living cells and a natural extracellular matrix with a synthetic Dacron support, it still failed due to clotting.
Cell-seeded conduits have been tried that require resorbable, non-biological scaffolds to generate sufficient mechanical strength. In this approach, cells (typically smooth muscle cells) are seeded into tubular structures made from materials such as polylactic acid. These vessels are susceptible to the same thrombosis/inflammation failures associated with an immune response. It is also difficult to exactly match their biomaterial degradation rates with tissue remodeling. Vascular grafts made from synthetic materials also are prone to bacterial colonization and infection, which can result in loss of function or serious systemic complications.
Furthermore, these vessels provide no mechanism to limit the proliferation of smooth muscle cells. Smooth muscle proliferation may infiltrate the lumen of the vessel and occlude it in a process called intimal hyperplasia. Finally, synthetic grafts have very different mechanical properties from natural tissues. These differences, particularly in tissue compliance, may induce adverse remodeling responses and graft failure due to localized non-physiological hemodynamic forces.
One promising solution to the problems associated with synthetic-based vascular grafts is to assemble blood vessels in vitro using only the patient's own cells and then re-implant them into the patient. This approach is called tissue engineering. In theory, tissue-engineered blood vessels (TEBVs) should provide mechanically stable vessels built only from autologous tissue, therefore generating no immune responses. Another advantage to a tissue engineering approach is the ability to manipulate the vessel ends to facilitate grafting of the vessel into place. Tissue engineering has been used successfully in the past to build two-dimensional structures such as skin, but has had only limited success with three dimensional tissues and organs such as TEBVs. The most common problem with three-dimensional engineered tissues is a lack of structural integrity and mechanical strength. This is a particular problem for TEBVs, since these vessels will be subjected to significant mechanical loads both from blood pressure (which may be abnormally high in patients with heart disease), as well as relative motion between the anchoring sites of the vessel (the aorta and the myocardium). Moreover, the TEBVs must demonstrate sufficient suturability and tear-resistance to allow surgical handling and implantation.
Recently, a tissue engineering technique has been developed that is a radical departure from prior art techniques. This work is described in U.S. Pat. No. 5,618,718 and in an article entitled “A Completely Biological Tissue-Engineered Human Blood Vessel,” L'Heureux, N., et al., FASEB J. 12:47–56, 1998, both of which are incorporated herein by reference. A fully biological and autologous human TEBV, with no synthetic materials, was made and found capable of withstanding physiological burst pressures in excess of 2000 mm Hg. These vessels were suturable and maintained patency for one week when xenografted into a dog. A living graft of this type is self-renewing with an inherent healing potential. The completely biological graft can be remodeled by the body according to the demands of the local mechanical environment. Moreover, the absence of synthetic materials precludes foreign body reactions, thus increasing the likelihood of long-term graft success.
These prior art TEBVs are prepared by rolling sheets of cultured fibroblasts and smooth muscle cells around a tubular mandrel. After a 2–8 week maturation period, the mandrel is removed and the vessel is seeded with endothelial cells. In this prior art procedure, the smooth muscle cells provide the TEBV with the ability to emulate a real blood vessel's ability to expand and contract. The smooth muscle cells also are responsible for the superior burst strength which in the past had been provided by the synthetic materials or scaffolds.
Even these prior art fully autologous TEBVs have significant problems. One of the biggest problems with TEBVs made from smooth muscle tissue is the difficulty associated with detaching and rolling the sheets of tissue while maintaining uniform vessel thickness. The inclusion of a smooth muscle layer complicates and lengthens the overall fabrication time by at least three weeks. Moreover, smooth muscle cells tend to uncontrollably proliferate. This undesired proliferation of cells may occlude the vessel. Moreover, the prior art rolling and tissue fabrication processes need to be improved and automated in order to obtain a clinically viable TEBV required for bypass grafts.
Contrary to the expectations in the art, a mechanically stable and fully autologous TEBV structure was discovered which could be fabricated without using smooth muscle tissue. The TEBV of this invention is made from fibroblasts and endothelial cells alone, thus eliminating the above-described problems caused by smooth muscle tissue. The new TEBV structure and method for its manufacture using an automated bioreactor are described below.